Spironolactone inhibits NADPH oxidase-induced oxidative stress and enhances eNOS in human endothelial cells.

Accumulating evidence indicates that aldosterone plays a critical role in the mediation of oxidative stress and vascular damage. NADPH oxidase has been recognized as a major source of oxidative stress in vasculature. However, the relation between NADPH oxidase in aldosterone-mediated oxidative stress in endothelial cells remains to be ascertained. The present study aimed to investigate the relevant role of NADPH oxidase in aldosterone induced oxidative stress and the functional consequence of this effect on endothelial function. Additionally, we attempted to examine the potential role of the mineralocorticoid receptor (MR) antagonist; spironolactone (spiro) in this scenario. Human umbilical artery endothelial cells (HUAECs) were incubated with aldosterone (100 nmol/L, 24 h) in the absence and presence of Spiro (1 µmol/L). The results showed that aldosterone significantly increased the protein expression of NADPH oxidase subunits (Nox2, p47(phox) and p22(phox)) and that spiro markedly inhibited these changes. Functionally, this was associated with an elevation in NADPH oxidase activity and 3-nitrotyrosine (3-NT) as a biochemical marker of oxidative stress. However, pre-incubation with spiro inhibited these consequences. Moreover, MR protein expression was upregulated by aldosterone whereas this effect was suppressed by Spiro. While aldosterone effectively inhibited endothelial nitric oxide (eNOS) protein expression, pretreatment with spiro markedly restored it to its normal level. In conclusion, the results achieved suggest that aldosterone may play a critical role in NADPH oxidase-mediated oxidative stress resulting in reduced eNOS expression in human endothelial cells. Spiro effectively reversed these consequences, suggesting its potential vasculoprotective effect in endothelial dysfunction.


Introduction
Oxidative stress contributes to increased cardiovascular morbidity and mortality associated with the activation of the reninangiotensin -aldosterone system. Studies in cultured cells in-vitro (1) and in rodent models in-vivo (2) demonstrated that aldosterone and/or mineralocorticoid receptor (MR) activation causes oxidative stress in addition to II, aldosterone can also cause endothelial dysfunction (3,4).
Several mechanisms account for the deleterious effects of aldosterone on endothelium with one important effect being a decrease in NO availability. This decrease may not only involve a reduction in NO production but also an increase in NO inactivation by reactive oxygen species (5,6). Aldosterone can increase oxidative stress by both increasing reactive oxygen species (ROS) production and reducing the ROS scavenging capacity of the cells. Aldosterone administration in uninephrectomized rats treated for 4 weeks with dietary 1% NaCl increased H 2 O 2 production by monocytes and lymphocytes (7). Similarly, aldosterone administration increased vascular superoxide production in normal rats (8). Aldosterone-induced ROS has been observed in different pathological situations in hypertensive animals (9) as well as in two different models of atherosclerosis (10).
On the other hand, several molecular sources of endothelial ROS formation have been suggested (11,12). NADPH oxidase complexes have been recognized as a major source of superoxide anions in endothelial cells (13)(14)(15). Thees NADPH oxidase complexes contain different catalytic NADPH oxidase subunits (16). Up to seven NADPH oxidase isoforms have been described in different cell types (17). The classical NADPH oxidase complex is composed 558 consisting of gp91 phox (Nox2) and p22 phox as well as cytosolic subunits (13,14). Besides the gp91 phox /Nox2-containing complex, Nox4 is the prominent isoform in endothelial cells (14,18). Nox2 is principally located in the endothelium and adventitia and it is essential for the activation of this enzyme in response to angiotensin II (14).
Although aldosterone plays a crucial role in vascular oxidative stress, its potential mechanism with respect to NADPH oxidase activation in endothelial cells still remains to be addressed. The present study was designed to identify the relevant role of NADPH oxidase in aldosteroneinduced oxidative stress and the functional consequence of this effect regarding endothelial function. Additionally, we attempted to examine the potential role of the MR antagonist; spironolactone (spiro) in this setting.

Cell Culture
All cell culture reagents and chemicals were purchased from Sigma Chemical Co., unless indicated otherwise. Primary cultures of HUAECs were isolated with collagenase IV and supplemented with 20% (v/v) calf serum, as were incubated with endothelium medium with a low calf serum 0.5% (v/v) for 24 h.These were subsequently treated with aldosterone (100 Estimation of NADPH oxidase activity NADPH oxidase activity was measured in cells by using lucigenin-derived chemiluminescence as described previously (20). The formation of a reactive oxygen species in response to aldosterone endothelial cells were detached, adjusted and incubated in white 96-well using trypsin (21). Subsequently, cells were transferred to a Krebs-3 , 2 PO 4 2 ·2H 2 4 ·7H 2 O, radical production was measured in the absence and reader (BMG, Offenburg, Germany). Results are expressed as relative light units per minute per 10 5 cells. In another set of experiments, the antioxidant properties of spiro were exa,ined using a xanthine/ xanthine oxidase assay. 500 xanthine oxidase in the absence and presence of a western lightening reagent. Then resulting chemiluminescence was measured in the absence and presence of a concentration-dependent (0.1, was performed using a membrane-permeable superoxide dismutase (SOD) mimetic.
Cells were lysed in ice-cold lysis buffer containing the miniprotease inhibitor cocktail, 1% Triton X-100, 0.1% SDS (Sodium Dodecyl Sulfate), 7.8. The protein concentration was determined using the BCA protein assay reagent (Perbio Science, Bonn, Germany). Equal amounts of membrane protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene Germany). After incubation in blocking solution (4% nonfat milk, Sigma), membranes were incubated with 1: 1000 Nox4, p22 phox , p47 phox and rabbit polyclonal antibody against MR (1:300) t 3-nitrotyrosine (3-NT) antibodies for 2 hours at room temperature. Membranes were washed and then incubated with a 1:3000 dilution of the second antibody (Amersham 1 h. The membranes were then detected with the enhanced chemiluminescence system (amersham life science). To correct for differences in protein loading, the membranes were washed and reprobed with a 1:1000 dilution of monoclonal antibody to human ß-actin (abcam). The relative intensities of the protein bands were analyzed by a scanner (model Scanmaker 8700, Microtek

Statistical analysis
Data are expressed as means ± SEM. A oneway analysis of variance (ANOVA) followed by Tukey's post test was used to assess the presence statistical analyses were accomplished, using the computer software GraphPad Prism 4 for Windows (GraphPad Software, USA).

Effect of aldosterone on NADPH Oxidase, 3-NT and eNOS expressions
HUAECs stimulated by aldosterone upregulation in NADPH oxidase, Nox2 (p < 0.01, Figure 1A) as well as the cytosolic p47 phox (p < 0.05, Figure 1B) and membrane subunits p22 phox (p < 0.05, Figure 1C) protein levels in comparison with the control. Pretreatment inhibited these changes. Aldosterone did not protein level. Moreover, stimulation of HUAECs 3-NT protein level as a marker of oxidative 2). On the other hand, aldosterone was found to exhibit a marked downregulation in eNOS 0.01) restored it to its normal level ( Figure 3). Furthermore, preincubation of the HUAECs with aldosterone resulted in an upregulation of MR this response ( Figure 4).

Effect of aldosterone on the NADPH oxidase activity
The stimulation of the HUAECs with increase in NADPH oxidase activity as measured by lucigenin-derived chemiluminescence (p < 0.001). However, pretreatment with Spiro (1 effect ( Figure 5A). It is noteworthy that the increase in superoxide anion production measured by lucigenin-mediated chemiluminescence was the xanthine oxidase inhibitor nor the uncoupled eNOS inhibitor was able to inhibit the lucigenin enhanced chemiluminescence. SOD was able to completely inhibit the lucigenin-enhanced chemiluminescence, indicating that these chemiluminescences are superoxide aniondependent. Regarding testing the antioxidant properties of Spiro using xanthine and the xanthine oxidase assay, spiro could not inhibit the suproxide anions generated by xanthine and the xanthine oxidase system. ( Figure 5B).

Discussion
The present study showed that stimulation of the HUAECs with aldosterone resulted in expression and activity. These changes were inhibited by the MR antagonist, spiro. We used a approximation to the in vivo situation, particularly Quantitative analysis of Nox2 protein expression, normalized with -actin, by scanning densitometry. (B) Aldosterone induced the p47 phox protein level, this was inhibited on pre-treatment with spiro. Quantitative analysis of the p47 phox protein, normalized with -actin, respectively, by scanning densitometry. (C) Preincubation of HUACEs with aldosterone resulted in the upregulation of inp22 phox protein expression and this was inhibited on pre-treatment with spiro. Quantitative analysis of the p22 phox protein, normalized with -actin, respectively, by scanning densitometry. Values for each bar are mean ± SEM from 4 separate experiments and expressed as % of control, p < 0.05, p < 0.01 versus control or indicated bar. Spiro: Spironolactone.

Control
Aldosrerone Aldosrerone + Spiro   In contrast, the NADPH oxidase inhibitor, apocynin reduced ROS formation supporting the hypothesis that NADPH oxidases are indeed a major source of vascular oxidative stress in our system model. Previous studies have suggested that ROS produced by NADPH oxidase mediate many angiotensin II effects in the cardiovascular system (14, 26). Several reports suggest an important role for aldosterone in the regulation of NADPH oxidase. Recently, it has been reported that aldosterone increases NADPH oxidase expression in the vasculature (28). Systemic administration of aldosterone increases oxidative stress in the heart, vasculature, kidney and increases macrophage NADPH oxidase (29). In addition, MR activation contributes to angiotensin II-mediated activation of NADPH oxidase in the heart and aorta (30). Furthermore, exogenous aldosterone stimulates aortic expression of p22 phox and Nox2 through an MR-dependent mechanism and of p47 phox mRNA through both an angiotensin type 1 receptor and MR-dependent mechanisms (31). In this regard, the present study demonstrates a marked upregulation of Nox2, in contrast to Nox4 that did not exhibit any change. Accordingly, Nox2 might be considered as the relevant isoform involved in aldosteronemediated NADPH oxidase activation. On the other hand, the cytosolic component of the p47 phox component was shown to have a pivotal role in the regulation of enzymatic activity. It has been reported that the hypertensive response and production of vascular superoxide was markedly blunted in p47 phox knockout mice (32). It has been also shown that aldosterone induced NADPH oxidase activation and membranous translocation of p47 phox in HUAECs (33). In this context, the current study shows that aldosterone increased the p47 phox protein level. Thus, aldosterone can increase ROS production in HUAECs by activating NADPH oxidase via p47 phox translocational regulation. NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to a superoxide anion which can react with nitric oxide to form short-lived peroxynitrite. Peroxynitrite forms stable 3-NT-conjugated protein moieties (24). In this context, aldosterone was found to increase in the 3NT content as a biochemical marker inhibited with pretreatment using spiro, supporting a potential role of MR in oxidative stress. Spiro was able to inhibit aldosteronemediated NADPH oxidase activation, suggesting MR might be considered as upstream of NADPH of the xanthine/xanthine oxidase assay, where study demonstrates that spiro can antagonize aldosterone-mediated superoxide production and this is attributable to its inhibition of the NADPH oxidase enzyme. It has been reported that superoxide production reduces nitric oxide bioactivity while reducing the expression of nitric oxide synthase (34). Evidence of endothelial dysfunction was seen in isolated renal artery segments and aortic rings from rats exposed to a model of excessive MR stimulation. In animals treated with spiro, normal endothelial function was restored (8,35). Similarly, in rabbits fed a proatherosclerotic diet, treatment with spiro normalized superoxide formation and improved endothelial function (36). In healthy male volunteers, aldosterone has been shown to cause acute endothelial dysfunction (37). the inhibitory effect of aldosterone on eNOS expression. Importantly, using MR-reactive antibodies and western blotting we investigated MR protein expression in the HUAECs in response to aldosterone. This suggests that MR was the main receptor mediating the pro-oxidative effect of aldosterone in the current investigations.
the previous in-vivo study demonstrating induces an increase in NADPH oxidase activity measured by lucigenin-enhanced chemiluminescence and this was markedly inhibited by of spiro. Spiro could not inhibit the suproxide production generated by the xanthine and xanthine oxidase system. Values for each bar are mean ± SEM from 8 separate experiments and expressed as % of control p < 0.01 and p < 0.001 versus control or as indicated bar. Spiro: Spironolactone.
administration to hypercholesterolemic rabbits normalized superoxide generation, decreased NADPH oxidase activity to basal levels and nearly normalized endothelium-dependent vasorelaxation.(10). A recent study showed similar inhibition of atherosclerosis when eplerenone reduced markers of oxidative stress including the ability of macrophages to oxidize low density lipoprotein, macrophage superoxide anion release and the susceptibility of low density lipoprotein to oxidation (38). Regarding the mechanism of action aldosterone, it has been reported that aldosterone binds intracellular MR and translocates it to the nucleus, where it binds to its ligand and interacts with the regulatory region of target gene promoters (39). In contrast, aldosterone might have a non-genomic effect within minutes (40). Importantly, we could not detect ROS production within 2 h of stimulation by aldosterone, it took much longer as was previously reported (28, 35). However, some reports showed that aldosterone induces ROS production through the activation of NADPH oxidase within 30 min (41,42). Such a discrepancy might be attributed to using different cell types or different experimental conditions.
In conclusion, the present results demonstrate that aldosterone stimulates NADPH oxidasemediated oxidative stress thereby reducing eNOS expression. In addition, the MR antagonist, spiro appears to play a potential role in inhibiting these consequences. Thus, the current study suggests that NADPH oxidase might act as key regulator in aldosterone--mediated oxidative stress, thereby reducing eNOS expression. This study adds a new dimension to the understanding of the role of aldosterone in the activation of NADPH oxidase in human endothelial cells and supports the notion that aldosterone can induce the dysregulation of endothelial cells. This knowledge may lead to novel strategies for the prevention of oxidative stress and endothelial dysfunction via the blocking of MR.

Acknowledgment
I am grateful to all of the technicians in Prof. Morawietz 's lab (Faculty of Medicine, Dresden University, Germany) for their technical assistance in cell culture technology and their kind help in achieving this work.

Source of Funding
This study was funded by DAAD (German Ministry of higher education of Egypt.